This revised, competitive renewal application is based on the observation that medically important protists make truncated Asn-linked (N-glycan) precursors, which contain 1-2 sugars (Giardia and Plasmodium) or 7 sugars (Entamoeba and Trichomonas) rather than the 14 sugars of most animals and fungi. This remarkable finding, which was made in collaboration with my colleague John Samuelson, who is an expert in parasitic protists, has numerous important and exciting consequences. First, while the truncated N-glycan precursors of Entamoeba and Trichomonas are sufficient for N- glycan-dependent quality control (QC) of protein folding, N-glycan-dependent QC is absent from Giardia and Plasmodium. Second, there is positive selection for N-linked glycosylation in eukaryotes with N- glycan-dependent QC. Third, truncated N-glycans of the protists affect the substrate specificity of the oligosaccharyltransferase (OST) that transfers N-glycans from the lipid precursor to the nascent peptide. Fourth, wheat germ agglutinin and Concanavalin A affinity chromatography dramatically enriches the N-glycomes (glycoproteins with N-glycans composed of GlcNAc2) of Giardia and Entamoeba, respectively. Fifth, Giardia has a single nucleotide sugar transporter (NSTs) for UDP-GlcNAc, such that this protist may be used as an alternative to Saccharomyces for testing NSTs. Sixth, Entamoeba and Trichomonas each have unprocessed N-glycans on their surface, which are the target of anti-retroviral lectins. Seventh, Entamoeba makes unique complex N-glycans, in which 1-1,2-linked Gal and then poly- 1,3-linked Glc are added to both arms of biantennary GlcNAcMan3. Eighth, because the N-glycan precursor of Trichomonas lacks Glc, it is puzzling that its membranes have 200 times the dolichol phosphate glucose (Dol-P-Glc) synthase activity of yeast or mammalian membranes. Preliminary studies suggest Trichomonas uses Dol-P-Glc for O-linked glycosylation and for synthesis of novel glycolipids. In light of these and other results, three Specific Aims attempt to answer the following questions: Specific Aim 1. What are the functions of the very short (GlcNAc and GlcNAc2) N-glycans of Plasmodium and Giardia (40% of the effort)? How do the short N-glycans of these protists affect their OSTs? What glycoproteins compose the Giardia and Plasmodium N-glycome? What is the effect of inhibiting or deleting N-glycosylation in these organisms? Aim 2. What are the functions of the short N-glycan precursors of Entamoeba and Trichomonas (40% of effort)? What are the final N-glycans of Trichomonas, which has glycosyltransferases absent in other protists? What glycoproteins compose the Entamoeba and Trichomonas N-glycome? Can we use anti-retroviral lectins to target the unprocessed N-glycans of Entamoeba and Trichomonas? Aim 3. What are the novel uses of Dol-P-Glc and Dol-P-Man by Trichomonas (20% of effort)? Are Trichomonas homologs of yeast PMTs and mammalian POMTs involved in synthesis of O-glycans from Dol-P-Glc and Dol-P-Man? What are the glycolipid products from these substrates? Lay description: Addition of sugars to proteins is essential for all organisms, and deficiencies in protein glycosylation are associated with human genetic diseases. The focus here is on sugars added to parasites that cause malaria, dysentery, diarrhea, and vaginitis, with the goals of understanding better how these organisms cause disease and how protein glycosylation works in all cells.